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a. Principle. Nonnucleated immature erythrocytes contain nuclear remnants of RNA and the cell is known as a reticulocyte. To detect the presence of this RNA, the red cells must be stained while they are still living. This process is called supravital staining. With supravital staining, the RNA appears as a reticulum within the red cell.

b. Reagent.

(1) New Methylene Blue Solution. Dissolve 0.5 grams of new methylene blue, 1.4 grams of potassium oxalate, and 0.8 grams of sodium chloride in distilled water. Dilute to 100 ml. Filter before use.

(2) Brilliant Cresyl Blue Solution. Dissolve 1.0 grams of brilliant cresyl blue in 99 ml of .85 per cent sodium chloride. Filter before use.

c. Procedure.

(1) Place 3 or 4 drops of new methylene blue and 3 or 4 drops of blood (venous or capillary) in a small test tube.

(2) Mix the tube contents and allow to stand for a minimum of 15 minutes. This allows the reticulocytes adequate time to take up the stain.

(3) At the end of 15 minutes, mix the contents of the tube well.

(4) Place a small drop of the mixture on a clean glass slide and prepare a thin smear.

(5) Counterstain with Wright's stain, if desired.

(6) Allow smear to air-dry.

(7) Place the slide on the microscope stage and, using the low power objective, locate the thin portion of the smear in which the red cells are evenly distributed and are not touching each other.

(8) Switch to oil immersion magnification and count the number of reticulocytes in 5 fields of 200 RBCs.

d. Calculation.

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e. Sources of Error.

(1) Equal volumes of blood and stain give optimum staining conditions. An excess of blood causes the reticulum to understain. An excess of stain usually obscures the reticulum.

(2) Crenated erythrocytes and rouleaux formation make an accurate count difficult to perform.

(3) Stain precipitated on erythrocytes causes them to appear as reticulocytes

(4) Dirty slides cause uneven spreading.

(5) The dye solution should have adequate time to penetrate the cell and stain the reticulum.

f. Discussion.

(1) Reticulocytes are nonnucleated erythrocytes that exhibit blue reticulum strands within their cytoplasm when stained supravitally. When stained only with Wright's stain, they are buff-pink in color and larger and darker than erythrocytes.

(2) Reticulocytes serve as an index of the activity of the bone marrow in blood regeneration. As such, these counts are of value in following anti-anemia therapy. Satisfactory response to therapy is evidenced by an increase of reticulocytes in the peripheral blood. Increased reticulocyte counts also occur whenever there is rapid bone marrow activity as in leukemia or blood regeneration associated with hemorrhage or hemolysis. Decreased reticulocyte counts occur in conditions in which the bone marrow is not producing adequate red blood cells, such as aplastic anemia.

(3) Several methods for staining and counting reticulocytes are in common use. Compared to the use of alcoholic solutions of dye, methods employing saline solutions of new methylene blue can give slightly higher values for reticulocytes. For comparative studies, the same method should be used throughout the work.

(4) Precipitated stain is often confused with reticulum but can be recognized by its presence throughout the smear and apart from the red cells. Precipitation can be eliminated as a source of error by frequently filtering the stain.

(5) An alternate method of counting reticulocytes utilizes the Miller disk that is placed inside the microscope eyepiece. This disc consists of 2 squares as shown below in figure 5-2. The area of the smaller square (B) is a tenth that of square A. Therefore, if there are 40 red cells in square A, there should be four red cells present in square B. When employing this method to count reticulocytes, the red cells in square B are counted in successive fields on the slide, until a total of 500 red cells have been counted. At the same time, the reticulocytes in square A are enumerated. At the completion of the count, theoretically, the reticulocytes obtained in this way are divided by 50, in order to obtain the percent reticulocytes present in the blood.

Figure 5-2. Miller disc.

g. Normal Values.

(1) Birth. Two and one-half to 6.0 percent, but falls to adult range by the end of second week of life.

(2) Adults (both sexes). Five-tenths to 1.5 percent.

Curriculum design: David L. Heiserman
Publisher: SweetHaven Publishing Services

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